Bile is the principal pathway for cholesterol elimination from the body, and high density lipoproteins (HDL) in plasma are the principal source of biliary cholesterol. However, hepatocellular mechanisms for uptake, transport and biliary secretion of HDL-derived cholesterol remain poorly understood. Scavenger receptor class B type I (SR-BI) is highly expressed in liver and promotes uptake of HDL lipids. Caveolae are cholesterol- and sphingolipid-rich membrane microdomains, which participate in cellular cholesterol transport. Both caveolin-1, the structural protein of caveolae, and SR-BI are expressed on the sinusoidal and canalicular domains of hepatocyte plasma membranes. Specific Aims 1 and 2 of this proposal will test the hypothesis that caveolae constitute a vesicular pathway that is responsible for sinusoidal HDL uptake and delivery to the canalicular membrane. Using mice with targeted disruption of genes encoding caveolin-1 and SR-BI, Specific Aim 1 will explore contributions of caveolin-1 and SR-BI to hepatic lipoprotein clearance and biliary lipid secretion. Primary cultures of hepatocytes from genetically engineered mice will be used to dissect contributions of caveolin-1 and SR-BI to binding and uptake of HDL. Specific Aim 2 will examine a role for caveolae in trafficking of HDL through the hepatocyte. Real time confocal fluorescence microscopy of HUH-7 hepatoma cells, which have been transfected with a green fluorescent protein-caveolin-1 fusion protein, will be employed to characterize uptake of luorescence-labeled HDL, processing within the endosomal compartment and trafficking to the canalicular membrane. An in vitro vesicle motility assay will be used to explore a role for microtubules in transport and sorting of vesicles containing HDL, SR-BI and caveolin-1. Hepatocellular secretion of phosphatidylcholines into blood and bile is critical for HDL metabolism and bile formation, respectively. Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein of unknown function that is highly expressed in liver. Specific Aim 3 will utilize PC-TP knockout mice to test the hypothesis that PCTP provides a nonvesicular pathway for phosphatidylcholine transport to the plasma membrane for incorporation into biliary vesicles and HDL particles. PC-TP knockout mice will be used to explore a role for this protein in biliary elimination of plasma cholesterol. These studies should elucidate the mechanisms of biliary lipid secretion at a fundamental cellular level and potentially lead to early interventions in cholestasis and cholelithiasis, as well as new strategies for management of hypercholesterolemia.